Coherent Raman scattering microscopy (abbreviated “CRSM”) has recently acquired considerable importance in image-producing chemical sample analysis, for example in biology, pharmacy, or food science. A variety of CRSM methods are utilized, for example stimulated Raman scattering (SRS), coherent anti-Stokes Raman scattering (CARS), and Raman-induced Kerr effect scattering (RIKES). The list of documents below will be referred to hereinafter regarding the existing art:    [1] Nandakumar, P., Kovalev, A., Volkmer, A.: “Vibrational imaging based on stimulated Raman scattering microscopy,” New Journal of Physics, 2009, 11, 033026.    [2] Freudiger, C. W., Roeffaers, M. B. J., Zhang, X., Saar, B. G., Min, W., Xie, X. S.: “Optical heterodyne-detected Raman-induced Kerr effect (OHD-RIKE) microscopy,” Journal of Physical Chemistry B, 2011, 115, 5574-5581.    [3] Saar, B. G., Freudiger, C. W., Reichman, J., Stanley, C. M., Holtom, G. R., Vie, X. S.: “Video-rate molecular imaging in vivo with stimulated Raman scattering,” Science, 2010, 330, 1368-1370.    [4] Mikhail N. Slipchenko, Robert A. Oglesbee, Delong Zhang, Wei Wu, Ji-Xin Cheng: “Heterodyne detected nonlinear optical imaging in a lock-in free manner,” J. Biophotonics, 2012, 5, 1-7.    [5] Zumbusch, A., Holtom, G. R., Xie, X. S.: “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett., 1999, 82, 4142-4145.    [6] Cheng, J. X. and Xie, X. S.: “Coherent anti-Stokes Raman scattering microscopy: Instrumentation, theory, and applications,” J. Phys. Chem. B, 2004, 108, 827-840.    [7] Evans, C. L. and Xie, X. S.: “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem., 2008, 1, 883-909.
In the CRSM technique, two pulsed light fields having pulse widths in a range from 100 fs to 20 ps, of different wavelengths, are directed through a confocal microscope optical system and focused onto the sample. The pulsed light fields are emitted onto the sample at frequencies that are typically in a range from 1 to 100 MHz. The light fields are spatially and temporally superimposed on one another on the sample via corresponding beam guidance and suitable focusing optics. In the SRS method or the image-producing superimposed RIKES method, for example, one of the two light fields is modulated in terms of intensity, frequency, or polarization at a specific frequency that is typically in the kHz to MHz range, before interacting in the sample with the other light field. For SRS and RIKES image production, the initially unmodulated light field is then sensed and, using a lock-in technique or envelope curve demodulation technique, the intensity modulation is extracted and presented in the form of an image. Reference is made to documents [1], [2], and [3] regarding implementation of the lock-in technique. The envelope curve demodulation technique is described in document [4]. In the case of CARS and CSRS a third light field is sensed as a consequence of interaction with the sample, and displayed as an image. This is described in documents [5], [6], and [7].
In all the CRSM techniques recited above, the measured signal is strong only if the difference between the frequencies of the incident light fields coincides with a vibrational resonance frequency in the sample. At present the best images in terms of spectral selectivity, signal intensity, and signal to noise ratio are obtained using picosecond laser light sources and optical parametric oscillators (OPOs) having pulse widths from 5 to 7 ps.
Among the various image-producing techniques based on coherent Raman scattering, the SRS image-producing method has attracted particular attention in the recent past because of the resonance-free background that is not present in the images. Because of the modulation and demodulation techniques explained above that are utilized in the SRS method, however, this method is also very sensitive in terms of the delay that must be established between the laser beams emitted from the two pulsed laser light source, in terms of the pulse synchronization that must exist during operation of the two pulsed laser light sources, and in terms of the time-related cyclical “jitter” that often occurs when the pulsed laser light sources are operated. A typical SRS image-producing device is moreover comparatively costly, and is limited with regard to its application capabilities.
It has therefore been proposed that in SRS imaging, the two pulsed laser light sources be replaced with continuous sources. In this case as well, one of the two laser beams is amplitude-modulated by means of a modulator. Although a solution of this kind is inexpensive, the resulting SRS measured signal is at least 106 times weaker than the measured signal obtained with the aid of pulsed laser light sources. It is evident from this that a device operating according to the SRS method with two continuous laser light sources is unsuitable for real-time image generation, in which approximately 25 individual images must be generated every second. In addition, continuous laser light sources adjustable over a broad wavelength band are in any case not yet readily available at this time.